GFP and its numerous related fluorescent proteins are now in widespread use as protein tagging agents (for review, see Verkhusha et al., 2003, GFP-like fluorescent proteins and chromoproteins of the class Anthozoa. In: Protein Structures: Kaleidescope of Structural Properties and Functions, Ch. 18, pp. 405-439, Research Signpost, Kerala, India). GFP-like proteins are an expanding family of homologous, 25-30 kDa polypeptides sharing a conserved 11 beta-strand “barrel” structure. The GFP-like protein family currently comprises well over 100 members, cloned from various Anthozoa and Hydrozoa species, and includes red, yellow and green fluorescent proteins and a variety of non-fluorescent chromoproteins. A wide variety of fluorescent protein labeling assays and kits are commercially available, encompassing a broad spectrum of GFP spectral variants and GFP-like fluorescent proteins, including DsRed and other red fluorescent proteins (Clontech, Palo Alto, Calif.; Amersham, Piscataway, N.J.).
However, the stability of fluorescent proteins is limited. Various approaches aimed at stabilizing fluorescent proteins have been undertaken. For example, Siemering et al. described the generation of a GFP mutant (GFPA) using site-directed mutagenesis, reporting that the mutant showed reduced sensitivity to temperature in both bacteria and yeast cultured at 37° C. (Siemering et al., 1996, Curr Biol 6: 1653). U.S. Pat. No. 6,414,119 described a GFP mutant showing modest improvements in thermal stability over wild type GFP (reportedly retaining fluorescence and solubility at 42° C., and showing some fluorescence at 50° C.). More recently, Pedelacq et al.,7 used directed evolution to increase the stability of GFP by selecting for resistance to the destabilizing effects of a poorly folding and aggregating ferritin sequence fused upstream. The first fusions were very weakly fluorescent, but with further evolution of the GFP, this external destabilization could be overcome and a variant (termed “superfolder GFP”) able to resist the folding interference of ferritin was selected. This was shown to be considerably more stable than standard GFP by a number of different measures, including resistance to thermal and chemical denaturation.
A number of different methods have been developed to create thermostable proteins, most of which involve the creation of libraries and the identification of improved proteins by selection or screening. Conceptually, the most straightforward way to identify proteins with improved thermostability has been to apply a thermal challenge to a collection of individual clones and test the remaining functionality of the clones, repeating this process if necessary, to combine useful mutations8-10. A similar method, which does not rely on such extensive screening requirements, involves direct selection of clones growing at elevated temperature within thermophilic bacteria. However, to date, this method has only been applied to the selection of thermophilic antibiotic resistance proteins11, 12, and as laboratory organisms typically do not grow at elevated temperatures, it has been difficult to generalize. As a result, considerable effort has been put into the development of alternative approaches which involve selection or screening for biophysical or biological properties which can serve as surrogates for, and are often correlated with, thermostability.
One of the first examples of this approach is the PROSIDE (protein stability increased by directed evolution)13-20 approach in which resistance to protease digestion is used as the surrogate property for protein stability, with filamentous phage infectivity being the selection modality. Proteins under test are expressed between two domains in g3p (the phage receptor for bacteria): if they are cleaved by protease, the filamentous phage loses the N terminal g3p domain and consequently its ability to infect; if the protein is protease resistant infectivity is maintained. This has been successfully used to increase the stability of the beta1 domain of protein G15, the cold shock protein of B. subtilis17 and ribonuclease T113. In another approach involving directed evolution, Shusta et al., showed that the display levels of heterologous proteins on the surface of yeast correlated with expression levels and thermal stability21, although exceptions to this have been recently described22.
Consensus engineering23, 24 is an approach to increase protein stability which does not use directed evolution, but the informational content of aligned sequences. By modifying a sequence so that it more closely resembles a consensus derived from the alignment of numerous proteins of a particular family, it has been found that significant increases in stability can be obtained. This has been applied to antibodies and antibody fragments5, 24-31, GroEL minichaperones32, 33, p5334, WW35 and SH3 domains36. More recently consensus engineering has been applied to the creation of novel proteins, rather than the stepwise modification of pre-existing ones to resemble a consensus. Perhaps the most striking success was the application to phytases37-40, in which a final protein with a Tm of 90.4° C. was obtained: 52° C. greater than the best component parental sequence40. Similar stability was obtained with a consensus ankyrin sequence based on the alignment of 2000 different ankyrins41-43. We recently applied this method to the creation of a consensus green protein (CGP)44.
Although we obtained a functional fluorescent protein, its Tm was 5° C. less than the monomeric Azami Green45 used to identify the sequences comprising the consensus. However, in this case no effort was made to examine the effects of individual mutations, and it is likely that some of the consensus mutations were destabilizing, as had been previously shown for the phytase37-40.
Other methods used to increase protein stability, relying heavily on structural information, include “helix capping”46-49 or optimization50-52, the introduction of salt bridges or their replacement by hydrophobic interactions53-59, the introduction of clusters of aromatic-aromatic interactions60-62 and rigidification strategies, in which disulfide bonds or glycine to alanine, or Xaa to proline changes are introduced63-65. However, most of these have been carried out on model structures, and none has been widely adopted.
Thermostabilization of proteins is regarded as important in a number of biotechnological and pharmaceutical applications. Within the context of industrial enzymes, thermostability leads to longer enzyme survival times, as well as more efficient reactions at higher temperatures and diminished microbial contamination, all of which result in diminished costs, while in the pharmaceutical arena, thermostability of protein therapeutics leads to longer half lives and more effective drugs1-3. Thermostability has also been regarded as important in the use of proteins as scaffolds to generate libraries of specific binders. It has been reasoned that if a starting scaffold is more stable, it will be more tolerant to the destabilizing effects of mutations, or insertions, used to mediate binding. This has been shown for affinity reagents based on ankyrins4, and has also been applied to the creation of phage antibody libraries5. Finally, proteins of increased thermostability are more resistant to mutations than the protein from which they are derived, promoting evolvability by providing greater permissivity to mutations leading to novel functions6, 7.